Culture method for highly expressing erythropoietin in serum-free medium and cho cells
A technology of serum-free medium and culture method, which is applied to the culture field of high-efficiency expression of erythropoietin, can solve the problem of not being able to efficiently and stably express EPO in CHO cells, and is suitable for large-scale production, improves the degree of glycosylation, and has stable expression. Effect
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Embodiment 1
[0028] According to the culture process, the seed cells were activated and amplified, then the cells were digested with trypsin, the cell suspension was prepared, inoculated, and after serum-containing culture was carried out in 10% bovine serum DMEM medium without MTX, the cells were separated in 0mmol / L sodium n-butyrate, 0.5mmol / L sodium n-butyrate, 1.0mmol / L sodium n-butyrate and 2.0mmol / L serum-free medium, the serum-free medium includes a ratio of 1:9 DMEM medium and CHO-S-SFM II medium. After culturing at 36.8±2° C. for 48 hours, the supernatants were collected, and the EPO expression levels in the culture supernatants were measured respectively. The results showed that 1-2mmol / L sodium n-butyrate can not only stabilize the expression of EPO per unit cell, but also have no obvious effect on the growth of cells. see results figure 2 .
Embodiment 2
[0030] According to the culture process, the seed cells were activated and amplified, then the cells were digested with trypsin, the cell suspension was prepared, and inoculated, the cells were added to 10% bovine serum DMEM with 0 μmol / L, 0.5 μmol / L and 1 μmol / L MTX respectively. After serum-containing culture was carried out in the culture medium, serum-free culture was carried out by adding 1.0mmol / L sodium n-butyrate to serum-free medium respectively, and the serum-free medium included DMEM medium and CHO-S-SFM II medium. After culturing at 36.8±2°C for 48 hours, the supernatants were collected, and the expression levels of EPO in the culture supernatants were measured respectively. The results showed that the addition of 0.5-1 μmol / L MTX to the medium could not only stabilize the expression of EPO per unit cell, but also had no obvious effect on the growth of the cells. see results image 3 .
Embodiment 3
[0032] According to the culture process, the seed cells were activated and amplified, then the cells were digested with trypsin, the cell suspension was prepared, inoculated, and after serum-containing culture in 10% bovine serum DMEM medium with 1 μmol / L MTX, the cells were separately Cultivate in serum-free medium without glycosylation reagent and glycosylation reagent containing D-galactose 250mg / L, D-mannose 250mg / L, N-acetylglucosamine 250mg / L, said serum-free The medium includes DMEM medium and CHO-S-SFM II medium with a ratio of 1:9. After culturing at 36.8±2° C. for 48 hours, the supernatants were collected, and the EPO expression levels in the culture supernatants were measured respectively. The results showed that glycosylation reagents could increase the proportion of sialic acid EPO in the culture supernatant. See the results of IEF+Western-blot Figure 4 .
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